The present invention is concerned with a process for the determination of fructosamine in body fluids by the reaction of a sample solution with a colour reagent and with enzymes having an oxidising action, as well as with a reagent suitable therefor.
Fructosamines are formed in blood from glucose present therein. The carbonyl group of the glucose reacts with free protein amino residues causing the formation of Schiff's bases. By means of an Amadori rearrangement, there then arise the fructosamines which have a stable ketoamino bond. The half life time of the fructosamine is, because of the stability of this ketoamino bond, practically identical with that of the serum proteins, the half life time of which is, on average, about 21 days. Since the extent of the fructosamine formation is proportional to blood glucose level, the fructosamine content gives an indication of sugar metabolism. The sugar metabolism must be continuously monitored, for example, in the case of diabetics. Since the blood glucose level is subject to considerable variation, its determination provides the physician with the metabolic position only at the time of sampling the blood. A further possibility for testing for sugar metabolism consists in the determination of the glycosilated haemoglobin (HbA.sub.1), this determination being suitable for a long-term monitoring of the sugar metabolism. However, in order now to have a medium term control, the measurement of the fructosamine content is very suitable since its half life time permits the metabolic control of diabetics by dieting and therapeutic measures over an average period of time of about 3 weeks. In combination with known clinical-diagnostic parameters, blood glucose, as well as glycosilated haemoglobin, by means of a serum fructosamine determination, there can now be provided a further dependable, specific and practical method for monitoring diabetics.
The hitherto known processes for the determination of fructosamine (for example Johnson et al., Clin. Chim. Acta, 127, 87-95/1982) depend upon the fact that fructosamine, which in an aqueous alkaline medium is present in the enol form and can easily be oxidised in this form, is reacted with an oxidation agent (colour-producing compound) which, in reduced form, is coloured, for example a tetrazolium salt. The formazan coloured material thereby formed can then be measured photometrically and is proportional to the amount of fructosamine.
However, this test principle has the disadvantage that, besides fructosamine, all easily oxidisable components present in the sample material, for example uric acid and bilirubin, or medicaments, for example .alpha.-methyldopa, and decomposition products of medicaments, as well as ascorbic acid, give rise to false measurement results since they also reduce the colour-providing compounds and cause formation of additional coloured material.
Furthermore, the known fructosamine determination processes are disturbed by the total amount of the protein content, which varies from sample to sample. This results in measurement value variations and thereby reduces the sensitivity of the determination process. These disturbances are known as matrix effects and appear particularly when additional proteins are added, such as is usually the case in the preparation of standard solutions. Thus, for example, increasing amounts of protein slow down the colour reaction with desoxymorpholinofructose (DMF) which is frequently added as fructosamine analogue in such standard solutions for calibration purposes.
Finally, a comparison with an HPLC reference method (see E. Schleicher et al., J. Clin. Chem. Clin. Biochem., 19, 81-87/1981) has shown that, with the known processes, there is obtained a high colour signal axis intercept, which corresponds to about 50% of the signal of an average, normal serum collective.
Further difficulties arise in the case of fructosamine determinations in hyperlipidaemic sera. In general, in order to be able to obtain a sufficiently large measurement signal even in the case of low fructosamine concentrations in a sample, it is necessary to use a ratio of sample to reagent of 0.1. In the case of excessive triglyceride concentrations, however, where there is a high proportion of sample material, the resulting turbidity of the test batch has a negative effect in the case of a photometric measurement. The fructosamine determination is then made considerably more difficult or even prevented.
In European Patent Specification No. 0215170, there is described a fructosamine determination using tetrazolium salts at pH 10 to 14 as an end point determination. Disturbances by ascorbic acid and glutathione are thereby overcome by the addition of strong bases, oxidation agents or enzymes, or by salting out. However, in this way, the disturbances due to lipaemic, icteric and uric acid-rich sera cannot be overcome. Furthermore, the carrying out of the test according to European Patent Specification No. 0215170 requires a time-consuming pre-reaction which cannot be integrated into most automatic anaylsers.
One of these difficulties was solved by a two-step process in which, in a first step, a neutral to acidic pH value is adjusted in the sample solution at which the fructosamine is present in the keto form and, therefore, practically cannot be oxidised. At this pH value, oxidising-acting enzymes are then added until the non-specifically reducing sample components have reacted away. The pH value is then increased into the alkaline range, whereby the fructosamine again passes over into its enol form and then the tetrazolium salt is added thereto which reacts with the fructosamine. In this way, the exactitude and the sensitivity of the known fructosamine processes could be very considerably improved.
It is an object of the present invention to provide a process which can be carried out in one step and permits a determination of fructosamine in body fluids which is at least as accurate and sensitive as that already achieved for the two-step process.